All-optical visualization of specific molecules in the ultrastructural context of brain tissue

Abstract

One aspect of the invention provides a method for preparing a biological sample. The method includes: exposing the biological sample to a photocrosslinkable substance adapted to bind to the biological sample, thereby forming a plurality of sample-photocrosslinkable-substance conjugates; embedding the sample in a hydrogel; and exposing the sample to light energy at a wavelength and intensity sufficient to cross-link the photocrosslinkable substance to the hydrogel.

1 . A method for preparing a biological sample, the method comprising:
 exposing the biological sample to a photocrosslinkable substance adapted to bind to the biological sample, thereby forming a plurality of sample-photocrosslinkable-substance conjugates;   embedding the sample in a hydrogel; and   exposing the sample to light energy at a wavelength and intensity sufficient to cross-link the photocrosslinkable substance to the hydrogel.   
     
     
         2 . The method of  claim 1 , wherein the photocrosslinkable substance is of X-Y-Z format wherein X can bind a biological substance, Y is a photocrosslinkable group, and Z is a reporter group. 
     
     
         3 . The method of  claim 1 , wherein the photocrosslinkable substance comprises diazirine. 
     
     
         4 . The method of  claim 1 , wherein the photocrosslinkable substance comprises one or more selected from the group consisting of: a reporter group, a Clickable reporter group, and an alkyne reporter group. 
     
     
         5 . The method of  claim 1 , wherein the light energy is UV light. 
     
     
         6 . The method of  claim 1 , wherein the light energy has a wavelength between 200 and 600 nm. 
     
     
         7 . The method of  claim 1 , wherein the hydrogel is swellable. 
     
     
         8 . The method of  claim 1  wherein the hydrogel is swellable and the hydrogel is modified by inserting additional molecules into the hydrogel to increase its size. 
     
     
         9 . The method of  claim 1 , wherein the sample is photocrosslinked after embedding in a hydrogel. 
     
     
         10 . The method of  claim 1 , wherein the sample is photocrosslinked before polymerization of the hydrogel. 
     
     
         11 . The method of  claim 1 , wherein the sample is photocrosslinked while being embedded in a hydrogel. 
     
     
         12 . The method of  claim 1 , wherein the sample is photocrosslinked to one or more selected from the group consisting of: a first and a second hydrogel network in a double-network hydrogel. 
     
     
         13 . The method of  claim 1 , wherein the sample is photocrosslinked to one or more selected from the group consisting of: a first and an nth hydrogel network in a multiple-network hydrogel, wherein n is a positive integer greater than  2 . 
     
     
         14 . The method of  claim 1 , wherein the photocrosslinkable substance binds to proteins. 
     
     
         15 . The method of  claim 1 , wherein the photocrosslinkable substance comprises an amine reactive group. 
     
     
         16 . The method of  claim 1 , wherein the photocrosslinkable substance comprises an NHS ester group. 
     
     
         17 . The method of  claim 1 , wherein the photocrosslinkable substance binds to nucleic acids. 
     
     
         18 . The method of  claim 1 , wherein the photocrosslinkable substance binds to lipids. 
     
     
         19 . The method of  claim 1 , wherein the photocrosslinkable substance comprises a lipid. 
     
     
         20 . The method of  claim 1 , wherein the photocrosslinkable substance binds to carbohydrates.